With an estimated one third of the world's population infected with Mycobacterium tuberculosis (Mtb) (i.e. more than two billion individuals) and 9 to 10 million new cases and 2 million deaths every year, tuberculosis (TB) is a global and worldwide health problem. Mycobacterium tuberculosis (Mtb) bacillus, the causative agent of TB, possesses a circular genome of 4 411 529 base pairs (bp) which was fully sequenced in 1998 (Cole et al., 1998, Nature 393: 537-44). Mtb encodes approximately 4000 genes; however the function and role in Mtb life cycle and pathogenesis of the majority of these genes have not yet been elucidated yet. It has been hypothesized for a long time that separate sets of genes are expressed during distinct and sequential infection phases, namely the active phase followed by the latent state and, when conditions are gathered, the resuscitation phase leading to a novel active phase. Recent evidence has shaken this classical dogma and the field is now acknowledging that a certain “leakiness” is taking place, i.e. expression of genes can happen in a phase-independent manner although to various thresholds. Moreover the latent nature of Mtb is also disputed: are bacteria mostly dormant, non-replicating, or do they continue to replicate and sometimes even escape from the infected cells into adjacent airways, thereby inducing recurring immune responses? (Ehlers et al., 2009, Infection 37: 87-95).
Generally, person-to-person transmission occurs by aerosolized droplets generated by a person suffering from pulmonary TB (active disease). Among those infected (an estimated 30% of exposed individuals), only 5-10% will develop active TB disease within 2 years post-exposure (known as primary TB). However, the majority of infected individuals develop latent infection (LTBI) which can last decades without clinical signs or symptoms of disease. LTBI represents a state of equilibrium in which the infected subject is able to control the infection but not completely eradicate the bacteria. Reactivation (active TB after remote infection) may occur at a later stage, particularly in the elderly or in immunocompromised individuals as in the case of HIV infection and treatment with TNF inhibitors. The risk of TB reactivation is estimated as 10% per lifetime and impaired immunity increases the risk to 10% per year.
There are several lines of evidence suggesting that stimulation of the cellular immune system plays a role in controlling TB disease (Rook et al. 2007, J Infect Dis 196:191-8). The central role of CD4 T lymphocytes to control the pathogen and prevent progression to disease (approximately 90% of Mtb infected subjects) is well established. For instance, HIV/AIDS patients with low CD4+ T cells count are more susceptible to progression to TB disease while antiviral treatments that elevate CD4+ T cells reduce progression to TB disease. However, CD4 T cells do not operate alone and are supported by CD8 T cells and other T cell subsets. In this respect, experience with tumor necrosis factor-alpha (TNFa) blockers and genetic polymorphisms such as interferon-gamma (IFNg) and other receptor deficiencies demonstrate the importance of specific cytokines and cytokine networks in controlling the disease, implicating the cellular immune nature of TB control in humans (Cooper et al., 2009, Annu Rev Immunol 27: 393-422).
The Mtb-caused million deaths every year are particularly dramatic considering that both vaccine (Bacille-Calmette-Guerin (BCG)) and antibiotics exist and are widely used. However, if BCG appears to be effective at preventing disease in newborns and toddlers, it does not protect adults and fails to prevent Mtb reactivation in latently infected persons. On the other hand, treatment of active TB with various antibiotic combinations appears efficacious but requires strong patient compliance with daily administrations of different drugs over several months. Moreover, while antibiotics are very efficient against wild type Mtb strains when taken properly, there is an alarming rate of appearance of drug resistant Mtb strains (e.g.“MultiDrug Resistant” (MDR), “eXtensively Drug-Resistant” (XDR) and “Totally Drug Resistant” (TDR) strains), mostly because of improper observance of this lengthy and costly drug regimen treatment. Development of effective TB vaccines is therefore a priority in this worrying context and two main approaches are being investigated for the last decade: replacement of BCG and BCG booster. More than a dozen vaccine candidates are now in clinical trials (for a review see Ottenhoff and Kaufmann, 2012, PLoS 8(5): e1002607). In addition, the field has also more recently considered using novel vaccine formulations to help in the treatment of Mtb infection, so called “therapeutic vaccines” to be used as novel stand-alone treatment or alternatively to adjunct to standard therapy, in particular for the treatment of drug resistant strains.
BCG replacement candidates aim at improving BCG efficacy and safety and are mainly based on live attenuated bacteria such as genetically modified BCG or Mtb strains engineered to express new sets of antigens that are absent from BCG or to overexpress Mtb antigens that BCG expresses but at a likely insufficient level or still to delete virulence genes and their regulators. Various recombinant BCG constructs have entered clinical trials to test their ability to substitute BCG. The most advanced VPM1002 currently in a Phase II trial is a urease-deficient rBCG that expresses the thiol-activated, cholesterol-binding listeriolysin (hly) from Listeria monocytogenes that has been shown to be safer than BCG in immunocompromised animals and to provide a superior protection in mice against challenge with Mtb (erode et al., 2005, J Clin Invest 115: 2472-9). Two additional rBCG have recently entered clinical assessment, respectively rBCG30 expressing Ag85B and AERAS422 expressing Ag85A, Ag85B and Rv3407 together with perfringolysin.
BCG boosters aim at inducing cellular and/or humoral immune responses and generally rely on recombinant vaccines designed for providing TB antigens, either as protein composition generally admixed with potent Th1-activating adjuvants or through viral expressing vectors, (for a review see Thaissa et al., 2010, Yale J. of Biol. and Medicine 83: 209-15; Andersen, 2007, Nature 5: 484 and Kaufman, 2012, Trend in Immunology 241: 1-7). Among the 4000 potential TB antigens, a number of them proved immunogenic in preclinical models.
One of the most advanced protein-based candidates is the hybrid 1 (H1) protein which consists of Ag85B fused to ESAT-6 (Langermans et al., 2005, Vaccine 23: 2740-50; Dietrich et al., 2007, J. Immunol. 178: 3721-30). A strong CD4+Th1 IFNg-mediated response was observed in humans when administered with IC31 adjuvant (Van Dissel et al., 2010, Vaccine 28: 3571-81). More recently, this vaccine was found to boost immune responses previously induced by either BCG or latent Mtb infection (Van Dissel, 2011, Vaccine 29: 2100-9). Another fusion protein Hyvac 4 (H4), which consists of Ag85B fused to the TB10.4 (Aagaard et al., PLoS One 4: 1-8) is in a parallel development program. The GSK's M72 fusion protein made of Rv1196 inserted in the middle of the serine protease Rv0125 showed a favourable clinical profile in terms of safety and immunogenicity when administered with different synthetic adjuvants (Von Eschen et al., 2009, Hum Vaccine 5: 475-82). One may also cite the so-called ID fusion proteins (WO2008/124647) such as ID83 made of Rv1813, Rv3620 and Rv2608 (Baldwin et al., 2009, Vaccine 27: 3063-71) and ID93 including Rv3619 fused to the three ID83 antigens (Bertholet et al., 2010, Sci Transl Med 2(53): 53ra74).
Viral-vectored TB vaccines that are being tested in clinical trials include the modified vaccinia virus Ankara (MVA) expressing the Ag85A antigen (MVA85A/Aeras-485; WO2006/72787), and the replication-deficient adenovirus (Ad) 35 expressing Ag85A, Ag85B and TB10.4 antigens (Crucell Ad35/Aeras-402; WO2006/053871). MVA85A has proved immunogenic in both naïve as well as BCG primed individuals, inducing high CD4+ T cell response (Mc Shane et al., 2004, Nat Med 10: 1240-4; Scriba et al., 2010, Eur J Immunol 40: 279-90) whereas Aeras-402 seemed to favor CD8 T cell and IFNg responses (Radosevic et al., 2007, Infect Immunol 75: 4105-15; Magalhaes et al., 2008, PLoS One 3, e3790; Abel et al., 2010 Am J Respir Crit Care Med 181: 1407-17).
More recent studies now focus on multi-phasic compositions (see e.g. WO2008/124647 and WO2011/144951). Some of these vaccine candidates have produced results in preclinical and clinical studies that demonstrate an ability to induce a robust cellular mediated immune response against Mtb (Thaissa et al., 2010, Yale J. of Biol. and Medicine 83: 209-15; Delogu et al., 2009, J Infect Developing Countries 3: 5-15). For example, the H56 fusion protein combining the latent Mtb Rv2660 together with the active Ag85B and ESAT-6 antigens showed potentially promising BCG booster activity although it has not yet reached clinical trials (Aagaard et al, 2011, Nature Med 17: 189-94; Lin et al., 2012, J Clin Invest 122: 303-14). However, these studies have highlighted the influence of various factors on the T cell response and protective efficacy such as the antigen doses (e.g. Aagaard et al., PLoS One 4: 1-8) and administration routes (Goonetilleke et al., 2003, J. Immunol. 171: 1602-9).
Tuberculosis is far from being controlled for different reasons: poor patient compliance with the prescribed standard-of-care in areas with limited resources, exacerbation of TB epidemics due to HIV coinfection, poor performance of BCG vaccination which is ineffective in protecting adults. In view of the increasing worldwide threat of TB and the inherent complexity of the Mtb infection and anti-mycobacterial immune response, there remains a need for improved vaccine strategies for diagnosing, preventing and treating tuberculosis, especially in endemic regions.
The present invention fulfils this and other needs by providing an immunogenic combination of Mtb antigens that is tailored for all phases of the natural course of infection.
This technical problem is solved by the provision of the embodiments as defined in the claims.
Other and further aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.